The overall goal of this program is to develop clinical immunoassays to report thrombolytically active tPA. tPA complexed to the main plasma inhibitor (plasminogen activator inhibitor-1; PAI-1) will not be detected. In Phase I, monoclonal antibodies with unique epitope specificity will be developed. Screening assays described in this proposal will verify reactivity with epitope expressed only on free tPA, the thrombolytically active form of the molecule. Lack of reactivity with tPA-PAI-I complexes will be documented. This precise specificity is critical as levels of PAI-1 may be extremely elevated during myocardial infarction causing significant amounts of infused tPA to circulate as enzymatically inactive drug. These antibodies will be further selected for genetic stability, affinity and avidity, reactivity with mutant tPA molecules and sensitivity and specificity in plasma. In Phase II, these antibodies will be used to develop ultra-rapid clinical assays which, for the first time, will allow the determination of the therapeutically optimal dose of tPA, will increase the efficacy and safety of thrombolytic therapy and will allow physicians to make decisions concerning modification of regimen of thrombolytic therapy.